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Biochimica et Biophysica Acta 1641 (2003) 121–135

Review

Revisiting the role of SNAREs in exocytosis and membrane fusion

Joseph A. Szule, Jens R. Coorssen*

Cellular and Molecular Neurobiology Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary,

Calgary, Alberta, Canada T2N 4N1

Received 8 November 2002; accepted 2 May 2003

Abstract

For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks

sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s)

of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis

begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the

roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other

critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the

pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often

prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function

upstream as modulators or priming factors of fusion.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Secretion; Exocytosis; Clostridial toxin; Lipid; Fusion pore; Molecular mechanism

1. Introduction Compartmentalization and the necessity of (regulated)

‘‘It is suggested that in BoTx poisoning the mechanism for

transmitter release has a reduced sensitivity to Ca, and

the level for activation by intracellular Ca is eleva-

ted. . .the release mechanism is in principle intact. . .’’Cull-Candy, Lundh and Thesleff (1976)

Although among the most fundamental and essential of

biological mechanisms, the molecular process of membrane

fusion, in both constitutive and triggered (regulated) release

events, still eludes our extensive attempts to dissect and

understand its underlying progression that results in the

merger of two separate and distinct biological membranes

and the compartments they previously delimited. This effi-

cient, targeted process constitutes the defining step in mech-

anisms as seemingly disparate as neurotransmission, wound

repair, hormone release, fertilization and blood coagulation.

However, might these release events perhaps be more similar

than they initially appear?

0167-4889/03/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0167-4889(03)00095-8

* Corresponding author. Tel.: +1-403-220-2422.

E-mail address: jcoorsse@ucalgary.ca (J.R. Coorssen).

fusion has been a conserved theme in biology. Considering

the extreme energy barriers inherent to the merger of two

distinct membranes, and the molecular rearrangements nec-

essary to accomplish leak-free coalescence of the apposed

membranes into a single continuous bilayer structure [1–5],

it is likely that fusion is a fundamentally conserved cellular

mechanism, and there is evidence to support this concept [6–

11]. It might be supposed that there had even been some

evolutionary processing in this regard, with different molec-

ular steps being optimized by selection until an energetically

efficient series of reactions was available. Subsequent cell

specialization would account for current differences in the

Ca2 + sensitivity and speed of various systems; testing and

selecting alternate Ca2 + sensors (different proteins and/or

isoforms of a given protein) and assorted accessory proteins

resulted in optimized functions in different cell types. The

resulting ‘variations on a theme’ likely consist of the same,

fundamentally conserved fusion mechanism [6–11] elabo-

rated upon with different accessory and modulatory compo-

nents to enhance efficiency, sensitivity to specific triggers

and localization to optimized membrane domains.

Although we take a reductionist approach in our ongoing

research directed toward dissecting the mechanisms under-

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135122

lying the Ca2 +-triggered membrane fusion step(s) of exo-

cytosis, we consider this from an inclusionist point of view;

work from a range of model systems (from yeast-to-inver-

tebrates-to-the-mammalian-CNS) contributes to the under-

standing of fundamental molecular processes. For example,

let us consider the role of ATP. Across several cell types, the

fusion step(s) of exocytosis has been shown to be indepen-

dent of ATP. In 1975, the ATP independence of Ca2 +-

triggered membrane fusion (of fully release-ready vesicles)

was first demonstrated in membrane preparations from

sea urchin eggs [12]. Subsequently, Baker and Whitaker

[13] extended this finding in experiments designed to

characterize the release mechanism. Elegant work from

others [14], studying regulated exocytosis in paramecium,

established that ATP was necessary for maintenance of a

primed release state. Holz et al. [15] confirmed these

findings in neuroendocrine cells, and identified an upstream

ATP-dependent priming stage; a neuroendocrine cell line

was used to identify priming factors [16,17]. The neuroen-

docrine cell studies also identified the rapid depriming of

vesicles (within 2–4 min) that occurs in such cell types

upon the depletion of ATP [15]. Time-resolved assays have

built on these findings to show that only a subpopulation of

morphologically docked vesicles are fully primed and

release-ready [18–20]. The ATP independence of synaptic

vesicle fusion has since been confirmed by Heidelberger et

al. [21]. Thus, before the originally proposed SNARE

hypothesis (suggesting that NSF-induced ATP hydrolysis

disrupts an inter-membrane complex and thereby induces

fusion [22,23]), it was known that ATP was required for

exocytosis, but not for the membrane fusion step(s).

As every system and every research approach has its own

inherent strengths and weaknesses as a ‘tool,’ we have tried

to refine our functional assays, indeed our interpretations, to

focus on the Ca2 +-triggered steps of membrane fusion. This

is particularly relevant to the current review in that much of

exocytosis research deals with ‘readouts’ of fusion rather

than direct measurements of the fusion step(s) itself. Caveats

to consider include (i) that we do not know all molecular

components, steps or events in the pathways of exocytosis

or fusion; and (ii) that most assays assess the pathway as a

whole or at best multiple molecular steps underlying a part

of the pathway. Thus, in our own work, we have found it

necessary to use a specific terminology in reference to

different pre-fusion, triggering and fusion events. We em-

ploy this terminology as an additional tool, to avoid seman-

tic and thus interpretational ambiguities; similar definitions

are broadly accepted in the field [24]. Thus, we define

membrane fusion as the full, non-leaky merger of two

previously distinct bilayer membranes into one continuous

bilayer via a ‘fusion pore;’ hemifusion refers to the merger

of only the proximal monolayers of two apposed bilayer

membranes. By extension, we consider the ‘fusogen,’ or the

‘fusion machine,’ to be that molecular entity directly re-

sponsible for the membrane merger event itself. By defini-

tion, all other molecular components and steps are

supportive or modulatory relative to the function of this

molecular entity. We also consider ‘pore expansion’ to be a

separate stage based on the existence of transient (‘kiss-and-

run’) fusion events [25–27] and evidence for specific

modulation of the expansion process [28–30]. Accordingly,

vesicle content extrusion after fusion may be yet another

mechanistically separable stage [31] or a simple physical

consequence of pore expansion and full (irreversible) merg-

er of the two fusing membranes.

Endocytosis, a multi-step fission process that recycles

membrane from the plasma membrane (PM), is the next

stage in the cyclic exocytotic pathway, but beyond the scope

of this review. In very general terms, endocytotic vesicles

pass either through the lysosomal system or rapidly re-enter

the small pool of actively recycling vesicles (f 10% of the

total vesicle population in a presynaptic bouton) [32,33].

Vesicles in the cytosol appear to exist in one or more

‘reserve’ populations that can be recruited to PM release

sites as required. Such vesicles must be appropriately

targeted and transported to sites on the PM. Although

possibly inter-related, we currently consider transport and

targeting to be effected by distinct, separable mechanisms.

Indeed, there appears to be much overlap of the subsequent

(or parallel) pre-fusion mechanisms that are broadly defined

as tethering, priming and docking. For the purposes of

working definitions, we describe tethering as an initial

stabilized contact between the vesicle and a targeted site

on the PM; this is a developing research focus [34,35].

Although likely to be multi-step processes with substantial

molecular and temporal overlap, we define priming and

docking as a potentially separable series of events. Here,

priming refers to any molecular reaction(s) that contributes

to optimization of the fusogenic potential of the vesicle, and

to its fully docked state; effective docking is required for the

most efficient translation of subsequent signals (‘triggers’)

for the fusion of the apposed vesicle and PM. As will be

described, SNARE interactions may thus be key (perhaps

culminating) events in priming. We consider ‘docked’ to be

the most fully optimized stage of inter-membrane contact

that can exist prior to actual fusion, and such vesicles

(forming only a very small proportion of vesicles that

appear morphologically attached to the PM) are often said

to be immediately ‘release-ready,’ awaiting only the neces-

sary trigger to initiate fusion (an appropriate increase in the

local [Ca2 +]free in many instances) [18,20,36]; we do not, at

the moment, subscribe to the idea that this represents a

hemi-fused state per se [37], but recognize that rapidly

passing through such a localized conformation is likely a

requisite molecular rearrangement on the molecular path-

way to fusion [38–40].

We stress that the actual molecular components essen-

tial (necessary and sufficient) to the stages described above

are as yet largely unknown, or at least unverified in terms

of definitive (but perhaps overlapping) roles in specific

steps of the overall exocytotic pathway. Indeed, at the

molecular level, many of these stages, particularly priming,

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135 123

docking, triggering and fusion, may well be rapid, tran-

sient, multi-step processes that are not easily dissected

experimentally. This emphasizes the fact that many estab-

lished assays actually assess multiple stages of the exocy-

totic pathway, and it is thus often impractical to claim

direct measure of fusion when either (i) changes in the

release of vesicular content are detected from (semi)intact

preparations; (ii) other stages in the pathway cannot be

directly and independently assessed; or (iii) the membranes

undergoing fusion cannot be directly verified. If interven-

tion does not change the assay outcome, fusion is unlikely

to have been affected, but if exocytosis is altered, this is

not evidence for a direct effect on the fusion step(s)

themselves.

2. An early tool: clostridial toxins

The clostridial neurotoxins have been among the most

important tools in understanding the exocytotic pathway.

Originally thought only to block neuronal function, this

selectivity of binding and uptake was eventually shown to

reside with the heavy chains of these toxins, while the

catalytic light chains (Zn2 +-dependent metalloproteinases)

are the actual active components that effect blockade of

neurotransmitter exocytosis [41]. This distinction led to

identification of the putatively selective substrates of these

toxins, the SNARE proteins [42,43]. Many VAMP/synapto-

brevin isoforms are cleaved by tetanus toxin (TeTx) and

botulinum toxin (BoTx) B, D, F and G, SNAP-25 isoforms

by BoTx A, C and E, and Syntaxins by BoTx C (reviewed

in Refs. [41,44]). Notably, mutations have resulted in at

least one cleavage resistant native isoform of each of the

three SNARE proteins (reviewed in Ref. [44]).

For well over a century, medical science has sought to

understand the actions of the clostridial neurotoxins. Many

of these now classical works highlight initial studies into

synaptic mechanisms and the vesicular hypothesis of neu-

rotransmission, thus charting the course of modern neuro-

physiology. Considering that the molecular targets and even

the proteolytic nature of the clostridial neurotoxins were

unknown at the time, these studies were elegantly insight-

ful. As early as 1939, Harvey [45] noted that spontaneous

release (muscle activity) still occurred following poisoning

with TeTx, and that repetitive stimulation (even by only

two effectively spaced stimuli) resulted in increased activ-

ity; increased intra-terminal [Ca2 +]free overcame the block

by TeTx. These findings were confirmed for preparations

poisoned with BoTxA [46], in which depolarization with

KCl was also shown to produce a transient recovery. The

work of Burgen et al. [46] was also important for extending

substantially earlier original observations that decreased

activity (of the test subject/preparation and thus of synaptic

activity) would slow the rate and extent of poisoning. These

observations suggest that synaptic activity (turnover in the

cyclic exocytotic pathway) enhances the effect of the toxins

and that the block must lie upstream of the fusion reaction

since enhanced stimulation can elicit some release (fusion),

albeit from a now rapidly exhaustible pool [47] (see also

Ref. [48]). Brooks [49,50] soon after concluded that

BoTxA did not directly inactivate the release mechanism

itself. Thesleff [51] also noted that doubling the external

(bath) [Ca2 +], adding TEA or inducing mechanical injury

to the motor-endplate overcame the block of release by

BoTx A. Spontaneous release events in denervated nerves

were also much less sensitive to BoTxA, again suggesting

that synaptic activity (vesicular turnover) promoted the

ability of the toxin to affect a block of neurotransmission

(see also Ref. [47]). Tetanic stimulation and reduced

temperatures were also shown to overcome blocks by both

BoTx A and TeTx [52].

Extensive studies by Parsons et al. [53], and by Harris

and Miledi [54], confirmed many of the findings described

above, illustrating [Ca2 +]-dependent recovery from TeTx

and BoTxD intoxication, respectively, by increased exter-

nal [Ca2 +], KCl-depolarization and paired stimuli. Both

studies reached conclusions of substantial long-term im-

portance to understanding synaptic function: toxin (i)

decreases the probability of transmitter release [53]; and

(ii) interferes with stimulus-secretion coupling [54,55].

More recent work on factors affecting SNARE complexes

has now reached similar conclusions regarding effects on

release probability [56,57], but the underlying mechanism

remains unknown. Notably, repetitive stimulation was also

shown to transiently relieve a complete block of neuro-

transmission produced by TeTx [58]. The most extensive

single analysis (even including a second dose of toxin)

may well be that by Cull-Candy et al. [59], who concluded

that the block by BoTxA affected the Ca2 + sensitivity of

the release mechanisms while clearly leaving the actual

fusion machinery intact. Thus, several decades of work

indicated that the target(s) of the clostridial neurotoxins

(then unknown) are not minimal essential components of

the native fusion machine, but rather that they are impor-

tant upstream modulators of synaptic efficacy. Subsequent

elegant studies also demonstrated distinct pre-fusion sites

of action of the different clostridial toxins, including effects

on priming [52,60–64]. However, caveats to be considered

include (i) the effectiveness and purity of some of the early

toxin isolates; (ii) the possibility that some nerve fibres

escape poisoning in the tissue preparations used; and (iii)

the potential presence of toxin-insensitive SNARE iso-

forms that might still function in a manner consistent with

the SNARE hypothesis. The first and second issues are

well addressed by the general consistency of findings,

using a variety of toxin and tissue preparations over several

decades, and by the range of toxin doses and poisoning

periods studied over this time. In addition, Cull-Candy et

al. [59] used second applications of toxin to ensure full

extent of poisoning. Gansel et al. [60] also used toxin

combinations, all without additional effect. However, the

potential presence of low levels of compensatory, toxin-

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135124

insensitive SNAREs cannot be easily ruled out in these or

other studies, yet recovery after extensive, long-term

blockade of exocytosis only occurs via the sprouting of

new terminals.

3. Comparative studies of toxin action

Despite the availability of the clostridial toxin light

chains, several factors have hampered the usefulness of

these toxins in terms of clearly defining the role(s) of the

SNARE proteins. These problems include the (i) existence

of toxin-insensitive SNARE isoforms, and (ii) lack of

routine, highly sensitive quantitative assays (to confirm

the extent of SNARE cleavage). These issues are further

complicated by the lack of selectivity of most assay systems

for specific steps in the exocytotic pathway; an observed

effect of these toxins in a given cell type clearly indicates a

role for the SNAREs in the dynamics of the cyclic exocy-

totic pathway, but tends to provide little additional evidence

as to the actual molecular contribution(s) of these proteins to

membrane fusion. While such experiments have been car-

ried out in a wide range of different constitutive and

regulated secretory cell types, blockade of release is rarely

consistent or complete [52,59,60,63–71]. While these stud-

ies clearly indicate strong evolutionary conservation of

molecular mechanisms involving SNAREs at one or more

stages of the exocytotic pathway, more definitive conclu-

sions are difficult. However, the ability to bypass clostridial

blockade clearly demonstrated that fusion mechanisms

remained intact and functional. Ca2 +- but not GTPgS-

triggered insulin release was blocked by TeTx and BoTx

B [72]. The BoTx A and C block of neurotransmission in

cultured hippocampal neurons could be partially overcome

by increased intra-terminal [Ca2 +]free, addition of cAMP or

by substituting Sr2 + for Ca2 + [68]. Fassio et al. [73] showed

that increased [Ca2 +]free (via ionophore) could bypass

inhibition of exocytosis by TeTx and BoTx F. Similar to

the early work on the neuromuscular junction, these studies

indicate that the final membrane fusion steps of excocytosis

are still functional after selective SNARE cleavage by the

different clostridial toxins, but that upstream regulation by

Ca2 + is perturbed. This conclusion is substantiated by work

in three very different systems.

First, despite extensive treatment with clostridial toxins

(singly or in combination) effecting the removal of the bulk

of the resident SNARE proteins from fully Ca2 + sensitive,

fusion-ready cortical vesicles (CV) isolated from unfertil-

ized sea urchin eggs [36,74,75], there was no effect on

Ca2 +-triggered homotypic fusion [76,77]. These findings

(1997–1998) were confirmed and extended using a variety

of coupled functional and biochemical assays, leading to the

conclusion that the SNARE complex might promote the

Ca2 + sensitivity of late triggered steps of exocytosis, but

was not an essential component of the minimal native fusion

machine [36,75–77]. Notably, fusion in the intact urchin

egg can be disrupted by clostridial toxins if the eggs are first

treated so as to un-dock CV from the PM, implying a more

likely role for the SNAREs in targeting and docking [78].

Second, studies focussing on the homotypic fusion of

isolated yeast vacuoles also indicated that SNAREs func-

tioned upstream of fusion; trans SNARE complexes

appeared to ‘activate’ a pathway to fusion, but the presence

of these complexes was not required for subsequent prog-

ress through the fusion pathway [79–82].

Third, detailed time-resolved analyses of Ca2 +-triggered

exocytosis from bovine chromaffin cells treated with differ-

ent clostridial toxins indicated inhibition of both the fast and

slow phases of exocytosis; BoTx A had the weakest effect

[70]. Inhibition of the slower phases of exocytosis implies

blockade of one or more steps upstream of fusion. Attempts

to use higher [Ca2 +]free to bypass the clostridial block were

confounded by a large release response that did not correlate

to dense core vesicles; elevated [Ca2 +]free are known to

trigger exocytosis of lysosomes in many cell types [83,84].

Nevertheless, there are several points to be considered in

interpreting these experiments. An ultra-fast phase of vesicle

release appeared still to be present in the toxin-treated cells;

the change in membrane capacitance upon triggering was

still equivalent to that measured for a small pool (f 10–35)

of fully release-ready vesicles [85–89]. A corresponding

amperometric signal appeared in some experiments but not

all [70]. The presence of this pool is somewhat surprising.

This small pool is most easily interpreted to represent fully

docked and release-ready vesicles, presumably with

SNARE proteins already complexed and thus inaccessible

to the clostridial toxins. However, observations of vesicle

dynamics near the PM indicate that even attached (‘docked’)

vesicles turn over within f 100 s [90]; pre-stimulus incu-

bations were for f 10 min in the experiments with clos-

tridial toxins [70]. If these vesicles were fully docked prior

to toxin delivery through the patch pipette, why did they not

dissociate from the membrane and become toxin sensitive

during the pre-stimulus incubation? If they did, how did

other vesicles, previously exposed to toxin, replace them?

One possible explanation is that many intact SNAREs might

still be operational in these experiments. There are clear

differences in the cleavage susceptibilities of recombinant

SNAREs in vitro and SNAREs in a native membrane. As

current measures of SNARE density on secretory vesicles

are f 10 times higher than estimated for the in vitro tests

used to assess toxin efficiency [70], substantial amounts of

functional SNARE proteins likely remained [91]. Then why

was exocytosis affected in the manner detected? Again, the

results are most simply interpreted to indicate that the

SNAREs function upstream of fusion, promoting the ‘nor-

mal’ physiological response because extensive secretion

was inhibited, but the actual fusion event was not (ultra-

fast phase). Combined with the findings described above,

from the experiments using CV and yeast vacuoles, the

results might be interpreted to indicate that once a group of

vesicles have gone through a trans SNARE ‘priming’ step,

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135 125

they, for some period of time, remain prepared to engage in

rapid fusion, and therefore represent a highly release-ready

pool. In some systems such as neuroendocrine cells, this

dynamic primed and immediate fusion-ready state might last

for f 2–4 min in the presence of cytosol, for up to 90 min

in yeast vacuoles, and for tens of hours (or longer; in the

absence of cytosol) in isolated CV [36,76]. This long-lived

fully primed and fusion competent (stage-specific) state of

docked CV, together with the Ca2 +-triggered disassembly of

SNARE complexes prior to fusion, has led to one possible

interpretation being that the SNAREs have not only carried

out their essential function(s) in exocytosis by this point, but

that their regulated removal from the site of inter-membrane

contact represents a critical step to ensuring fast, efficient

fusion [75].

A second study by Xu et al. [92] used an antibody (Fab

fragment) to SNAP-25, known to block SNARE complex

formation in vitro, to test the role of the complex in the

late Ca2 +-triggered steps of exocytosis. Again, both the

fast and slow phases of release were perturbed, but the

fusion machinery was clearly intact. Slowing the kinetics

of triggered release suggests a modulatory role for the

SNARE complex, consistent with an influence on the

probability of fusion in the physiological range of

[Ca2 +]free. The interpretation was that ‘loose’ inter-mem-

brane SNARE complexes were sufficient to support fusion

but that fully ‘zippered’ complexes were necessary for fast,

triggered fusion; these different states of the SNARE

complex may correlate with previously described differ-

ences in sensitivities to clostridial toxins [48]. However, as

SNARE complex formation in vitro has been suggested to

correlate with cis rather than trans complexes, and native

complexes were not assayed in this study [92], more

definitive statements concerning sites and effects of the

Fab fragment binding cannot be made. As with the earlier

experiments involving clostridial toxins, there is a question

as to why an incomplete block of the burst phase of release

occurs if fully docked vesicles turn over during the 10-min

intracellular exposure to the blocking antibody. Alterna-

tively, one might also postulate that binding of the Fab

fragment prevented effective clearance of SNARE com-

plexes thereby blocking fast, efficient fusion. However,

considering as a whole all the studies described above, and

noting that (i) the extent of inhibition of exocytosis by a

given clostridial toxin varies widely among different cell

types; and (ii) standard Western blotting or immunocyto-

chemical analyses are inherently insensitive, potentially

‘missing’ thousands or more copies of a given protein

(increased risk of ‘false-negative’ results) [91], firm con-

clusions as to the actual function(s) of the SNARE proteins

remained difficult. There was no way of confirming

whether the actual amounts of intact SNARE proteins

remaining after clostridial toxin treatments were sufficient

to account for the remaining function, whether SNARE

fragments acted to promote function [93,94], whether

residual function was due to the presence of toxin-insen-

sitive SNARE isoforms, or some combination of the above.

Additional clostridial toxin targets also could not be ruled

out [95–100].

As an alternative, molecular genetic approaches have

also been applied to the question of SNARE function. While

these studies have taught us a great deal concerning details

of protein–protein interactions, providing tools that will

undoubtedly prove invaluable to future mechanistic studies,

many of the same issues outlined above plague a definitive

interpretation of SNARE functions in membrane fusion.

While various SNARE mutations block or modify exocy-

tosis to varying extents, the assay formats used do not

permit detailed analyses of the Ca2 +-triggered fusion steps

themselves. Early SNARE mutations/knockouts in C. ele-

gans and Drosophila yielded results consistent with the

early clostridial toxin work in neuromuscular junction

preparations; fusion still occurred (spontaneous release),

but regulation of the release process was disrupted [101–

105]. Up-regulation of syntaxin 1A suggests a distinct role

in defining vesicles for the regulated secretory pathway

[106–108], and VAMP mutations suggest disruptions of

vesicle targeting and docking [109]. Washbourne et al. [110]

showed that SNAP-25 mutants incapable of binding VAMP

still support exocytosis. In addition, SNAP-25 mutations

that should disrupt SNARE complex formation and stability

still support normal exocytotic release from neuroendocrine

cells [111]. Most recently, knockouts of neural SNAP-25

[112] and VAMP2 [113] in mice (also showing perturbed

regulation of release) have been suggested to indicate that

SNAREs may not be essential to fusion. However, addi-

tional treatments with clostridial toxins were not performed

[114], and the presence of compensatory SNARE isoforms

cannot be ruled out. The evidence for such potential ‘rescue’

in vivo is clear [115–117]. Therefore, unless all the known

isoforms of a given SNARE in a specific organism can be

simultaneously knocked out, SNARE hypotheses remain

difficult to test even with the power of molecular genetics.

Given the critical role(s) of SNAREs in the exocytotic

pathway, even this approach would not provide a defin-

itive assessment as it would likely be fatal quite early in

development. Inducible knockouts are an obvious alterna-

tive, but still cannot address the issue of multiple SNARE

isoforms.

4. Fusion in vitro

One possible approach to testing SNARE hypotheses

requires the use of a stage-specific preparation such as the

urchin CV; by all available criteria [36,75,118], Ca2 +-trig-

gered CV–CV fusion in vitro, in the absence of cytoplasmic

factors, occurs through the same molecular pathway as

exocytotic release. Since cytoplasmic components, including

SNARE recruitment and assembly factors [119,120], are not

required for triggered CV fusion, an upstream modulatory

role (e.g. pre-fusion) for the SNAREs is suggested. Further-

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135126

more, despite extensive analysis by a number of laborato-

ries, evidence suggests CV membranes contain only one

isoform of each of the SNARE proteins [76,121]. Consid-

ering the sensitivity of both triggered fusion and the SNARE

proteins to proteases such as trypsin, it was anticipated that

treating isolated CV (thus exposing the entire complement

of vesicular membrane proteins) with broad spectrum pro-

teases might produce a ‘biochemical knockout’ of one or

more of the SNARE proteins [36]. If fusion persisted after

such treatments, a direct role for the SNAREs as essential

components of the native fusion machine would be ruled

out. This approach, utilizing stage-specific native mem-

branes, makes no assumptions as to which proteins are

important, and would theoretically result in complete

SNARE removal as all known SNARE isoforms (including

those insensitive to the clostridial toxins) have an exten-

sively conserved number of potential cleavage sites for

proteases such as trypsin. Indeed, the extensive cleavage

of SNAREs in vitro and in vivo has been demonstrated.

However, the effective analysis of a molecular mechanism

requires the accurate identification and quantification of

specific proteins. A quantitative, ultra-sensitive immuno-

blotting protocol has been developed and optimized [91].

Coupling sensitive detection with broad spectrum prote-

ase treatments has now permitted direct testing of SNARE

hypotheses. Quantitative removal of all three types of

SNARE proteins, in some cases including the complete

removal of syntaxin from CV, does not block Ca2 +-trig-

gered fusion [122,123]. Furthermore, despite substantial

ablation of the resident CV SNAREs (zf 90%), clostri-

pain treatments had no effect on the Ca2 + sensitivity or

extent of fusion or, perhaps most importantly, on the kinetics

of fusion. This is perhaps the most direct evidence that

SNAREs are unlikely to be essential components of the

minimal native fusion machine; if essential, such substantial

removal should most certainly have affected the kinetics of

Ca2 +-triggered fusion. Furthermore, the estimated energy

contributed by SNAREs and associated proteins (f 2 kT)

[123] is lower than that thought to be required to overcome

the hydration energy barrier at the membrane surface [124]

or for bilayer merger [3,5,125]. Differential protease effects

suggest the existence of a native fusion machine with an

inherently low Ca2 + sensitivity. One hypothesis is that

SNAREs and their immediate binding partners may act to

modulate the Ca2 + sensitivity of this native machine into the

physiological range of [Ca2 +]free [123].

The simplest interpretation of the above study is that

SNAREs are not essential components of the minimal native

fusion machine; there is little possibility of compensatory

proteins preserving function in the Ca2 +-triggered fusion

pathway of this particular stage-specific system. These find-

ings are consistent with work in the yeast vacuolar system

suggesting the existence of a fusion mechanism functioning

downstream of the SNAREs [81,82]. In addition, mutations

in genes related to fatty acid elongation and sphingolipid

synthesis bypass the need for vesicular SNAREs, suggesting

that SNAREs contribute to docking and the efficiency of

fusion, but not to the fusion mechanism per se [126].

Although not yet verified in other systems, these studies on

CV and yeast vacuoles support the concept of an upstream

‘priming’ role for the SNAREs, with alternate factors under-

lying the actual membrane merger steps. In contrast, other

work on constitutive exocytosis in yeast has led to an

alternate view of SNARE function, suggestive of a role in

fusion. In these studies, SNARE transmembrane regions

were replaced with covalently attached lipid moieties (ger-

anylgeranylated) and the slow constitutive pathway was

blocked; these results have been interpreted to suggest that

the transmembrane domain of SNAREs can function late in

fusion [127], an idea supported by studies demonstrating that

peptides corresponding to these transmembrane regions can

induce fusion of artificial membranes [128]. For reasons that

are not immediately apparent, these results are inconsistent

with the work on yeast vacuole fusion that indicates SNAREs

are not required at the fusion step [81,82]. Overall, both sets

of results in the yeast system appear inconsistent with the fact

that isolated CVand other native vesicles can fuse with pure

lipid target membranes [129–132]. While consistent with a

lipidic fusion pore, these results are more difficult to reconcile

with a proteinaceous, ‘channel-like’ fusion-pore, although

triggered conformational changes of a proteolipid could

perhaps contribute. Furthermore, recognized viral fusogenic

proteins, to which SNAREs have been compared, will also

catalyse merger with protein-free target membranes [133–

135]. This is most interesting when considering the saddle

model of hemagglutinin-induced fusion, where the viral

peptide inserts into its own membrane rather than the target

membrane [136]. SNAREs appear incapable of triggering

comparable events [137].

Thus, although hypotheses concerning transient inter-

membrane ‘‘SNAREpins’’ as fusion complexes are ques-

tionable in light of the results of studies in a number of

model systems, alternate hypotheses concerning the in-

volvement of SNARE transmembrane regions in hemifusion

intermediates require further investigation. However, it

remains unclear how these transmembrane regions would

converge in cases of cytosolic domain disruptions (that

obviate inter-membrane complex formation and function),

or in the absence of additional cytosolic factors that are

suggested to promote complex formation [119,120,138]. If

SNARE clearance from the fusion site is required for fast,

efficient fusion, convergence of these transmembrane

regions seems unlikely unless it contributes to localized

membrane destabilization well before the actual fusion

event, which might be promoted by specific membrane

domains [139].

5. Reconstitution

The strongest case for the SNAREs as fusogens has come

from the work of Weber et al. [137] who have used recom-

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135 127

binant SNAREs reconstituted into artificial lipid membrane

systems to study the effects of SNARE interactions in vitro.

This technically complicated experimental approach has

become an elegantly routine assay system in this group

[137,140–146]. The type of lipid vesicle preparation used

(small unilamellar liposomes) was originally developed to

retain small soluble content markers, the mixing of which

could be used to assess actual fusion events. Simply

assessing intermixing of membrane lipids was unsatisfacto-

ry as it did not represent complete fusion, and could even

occur by transbilayer exchange under conditions of close

inter-membrane apposition. One of the principal concerns

with interpretation of the reconstituted SNARE assays is

that this system has never demonstrated the capacity to

retain low molecular weight solutes; even large (f 5 kDa)

oligonucleotide markers showed a high level of ‘leakage’

[141]. But SNAREs are clearly capable of interacting to

yield complexes that could promote closer apposition of

membranes [147]. Although SNAREs may contribute, in

part, to overcoming the energy barrier imposed by the

ubiquitous hydration layer [1], and thereby perhaps promot-

ing nonspecific lipid exchange between the apposed mem-

branes, this state would be unlikely to spontaneously yield a

fusion site unless the bilayers were already destabilized.

Strong, nonselective increases in inter-membrane attractive

forces in vitro have indeed been shown to sufficiently

reduce inter-bilayer distances such that local dehydration

induces hemifusion [148,149]. It is unclear from the pub-

lished reconstitution assays the extent to which lipid ex-

change and bilayer mixing each contribute to the measured

signals, and to what extent the signals being assessed are

actually comparable to a native fusion event. Lipid bilayer

contact is required for bilayer mixing but not for nonspecific

lipid exchange. The structure of the SNARE complex [147]

suggests that an intervening hydrated interface of f 2nm

must remain between the SNARE-apposed membranes, thus

favouring nonspecific lipid exchange over true bilayer

mixing. This concern is reinforced when SNARE densities

are considered. Recent assessments reveal a striking simi-

larity between the SNARE densities measured on CV and

synaptic vesicle membranes [91]. These native SNARE

densities are 50-fold lower than required in the reconstituted

SNARE preparations [137]. Although a recent report indi-

cates that the in vitro assays will work with lower VAMP

densities, the VAMP was replaced with recombinant syn-

aptotagmin, which does not allay concerns regarding the

bilayer status (stability) of these preparations [142]. Clearly,

such a substantial increase in the local energy contributed by

SNAREs, relative to that in stable native membranes, could

well promote a nonspecific membrane merger event, but this

is unlikely to be representative of a biological fusion site. If

this is indeed what is occurring, it might, in part, explain the

rather slow fusion kinetics in these reconstituted systems

relative to triggered fusion in vivo. Although an increase in

[Ca2 +]free triggers fast fusion events in most secretory cell

types [18,20,150,151], reconstitution of synaptotagmin, the

putative essential Ca2 + sensor for triggered release does not

enhance fusion in the reconstituted preparations [142].

Similarly, lipid mixing between isolated synaptic vesicles

(containing synaptotagmin) and SNARE-containing lipo-

somes can be triggered by aggregation alone and is rela-

tively slow even at high [Ca2 +]free [152].

Over the last several decades, a substantial number of

proteins have been shown to promote aggregation, adhe-

sion, lipid mixing and even fusion in model membrane

systems [149,153–163]. In many cases, this fusion also

involves content mixing, is reasonably fast, and can be

triggered in some fashion that is at least reminiscent of

biological fusion events. Thus, there are clearly differences

between what can be demonstrated in vitro and actual

protein functions in vivo [164]. Although the native

cellular localization of the SNAREs may make these more

likely candidates than some of the other proteins shown to

promote fusion in vitro, this does not constitute evidence

that the results of assays in the reconstituted SNARE

preparations actually describe a minimal biological fusion

machine. Thus, although it has been argued that additional

factors may be present in native systems and that these

promote SNARE function, this is one possible interpreta-

tion, and not evidence supporting a role for the SNARE

complex as the minimal fusogenic entity. Indeed, the

SNARE interacting protein N-ethylmaleimide (NEM)-sen-

sitive factor (NSF), which is thought to mediate post-

fusion uncoupling of SNARE complexes and thus promote

their functions in vivo, has also been shown to indepen-

dently promote the fusion of liposomes [161,162,165]. It

has been argued that this effect of NSF is limited to

liposomes of specific lipid composition, and that since the

effect of the SNAREs extends to other lipid mixtures, the

SNARE complex represents a minimal fusogen [163].

However, liposomes are rarely used in an effort to mimic

global lipid compositions of entire cellular systems; rather,

they are most often used to mimic localized sites on

membranes. If a proteinaceous fusion machine (SNARE or

otherwise) has been evolutionarily conserved and opti-

mized, it seems unlikely that the same rationale does not

apply to the lipid components that form the bulk of the

membrane, and are the local substrates on/with which the

proteins must function. To postulate that one lipid mixture

is more likely or appropriate than another implies knowl-

edge of the actual lipidic species functioning at the native

fusion site. To the best of our knowledge, such specifics

remain unknown, although much elegant work has sug-

gested that certain lipid species are more likely than others

[129,131,132,159,165,166]. If optimization of the focal

lipid mixture at the fusion site is not critical, then SNARE

complexes at native densities [91] should also drive the full

fusion of membranes composed primarily of saturated, long

chain species of phosphatidylcholine, sphingomyelin or

ceramide; we are unaware of such a study. However, as

isolated CV are at a stage of fusion readiness that no longer

requires cytosolic factors, we suggest that NSF and other

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135128

identified promoters of SNARE function [119,120,167] are

more likely to act upstream of the triggered native fusion

event, and possibly also in later modulatory roles [168].

Thus, the reconstituted systems have demonstrated that the

SNAREs may code for some level of selectivity in regulating

the interactions of specific intracellular compartments

[169,170], and that the resulting inter-membrane complexes

may contribute to defining potential fusion sites. Therefore,

roles in targeting, docking and priming steps of exocytosis

are possible (e.g. pre-fusion), and disruptions at one or more

of these stages could explain the altered functions observed

when the cellular complement of SNAREs is targeted by

toxins, peptides, and so forth [59,70,92,171,172].

6. Biophysics of membrane fusion

Bilayer membrane fusion has been described in terms of

a progression of intermediate structures of lower free-

energy, which involve rearrangements of the lipid matrix.

Detailed mathematical modeling has been used to explore a

number of potential molecular-structural models; the se-

quence of structural rearrangements corresponding to the

lowest-free energy states has been identified as that involv-

ing a ‘stalk-pore’ transition [3,40,125,173,174]. The initial

step in this sequential rearrangement and merger of focally

apposed bilayer domains requires an input of energy to

overcome the hydration and molecular repulsive barriers

[1,2,175], thus bringing the apposed membranes into mo-

lecular contact. The subsequent rearrangements of the lipid

matrix reduce the net free-energy of the lipid assemblies,

mainly through the reduction of interstitial and curvature

energies. Interstitial energy can be reduced by hydrophobic

molecules occupying the interstitial volumes (within the

hydrophobic domain of the membrane) [176–179], or by

alternate packing and tilt of the acyl chains [5]. Curvature

energy is described in terms of spontaneous curvature,

where the curvature of a monolayer minimizes the bending

elastic free-energy [176]. Various lipid species influence the

spontaneous curvature of a membrane, either by adding

zero curvature energy, positive curvature energy (increased

propensity to forming curved assemblies with the polar

groups on the convex side) or negative curvature energy

(increased propensity to forming curved structures with

the polar groups on the concave side) [180]. Therefore,

the curvature properties of the lipids that compose fuso-

genic membranes influence the transition of intermediate

structures [39,181].

Briefly, the proximal (contacting) monolayers of the

apposed bilayer membranes merge, forming a contiguous,

highly curved ‘hourglass’ structure with a net negative

curvature (polar groups on the concave side), sandwiched

between the still intact distal (non-contacting) monolayers of

the apposed membranes (see Fig. 1 of Ref. [3], or Fig. 4 of

Ref. [181]); the result is an inter-membrane ‘stalk,’ which

defines a hemifusion state. Once formed, the stalk expands,

thinning until the distal monolayers merge, forming a lipidic

fusion pore which has a net positive curvature (polar groups

on the convex side). Recently, it was demonstrated that

model lipid assemblies undergo fusion through a process

that is consistent with stalk formation [182], and this has also

been seen in molecular dynamic simulations [183]. Exis-

tence of a stalk-like intermediate in native membrane fusion

is supported by the reversible block of a range of biological

fusion reactions by lysophospholipids [39,184,185]. This

block is downstream of SNARE function, and occurs irre-

spective of SNARE protein complexes functioning upstream

[76]. A lysophosphatidylcholine block was used to show that

SNARE interactions were not strict determinants of a docked

vesicle. SNAREs may function early in tethering/docking,

but their interactions are not essential to maintenance of the

fully docked and release-ready state [75]. Similar conclu-

sions have been reached using total internal reflection

fluorescence microscopy of TeTx and BoTx A expressing

chromaffin cells [186].

7. The Ca2+-triggered fusion steps of exocytosis

A decade of testing SNARE hypotheses has thus far not

provided the definitive experiment(s) to fully assess SNARE

contributions to exocytosis, although work on protease-

treated CV does indicate that SNAREs likely function

upstream of the (regulated) fusion steps [123]. Identification

of the SNAREs and a large number of interacting proteins

has provided a substantial catalogue of components neces-

sary for effective functioning of the exocytotic pathway.

Considering tests of function since the earliest neurophys-

iological analyses of clostridial toxin effects, we have

critically evaluated the most probable role(s) of the

SNAREs in the exocytotic pathway. Despite a range of

hypotheses, we currently view the SNAREs as modulatory

(promoting), working upstream of fusion and not represent-

ing minimal essential components of the native mechanism

directly responsible for the membrane merger events of

exocytotic fusion. Clearly, there are important implications

for current research directions, for mechanistic models of

exocytosis and for a molecular-level understanding of the

(regulated) fusion pathway.

While SNARE modulatory functions are clearly essential

to the physiology of exocytosis, perhaps in part defining

potential native fusion sites, there is clearly a difference

between contributions to the establishment and efficiency of

a mechanism, and the mechanism itself. We interpret the

SNAREs and associated proteins to function in targeting,

docking and priming, perhaps in the last priming step that

ensures full Ca2 + sensitivity, and thus a rapid, triggered

fusion response. Changes induced by SNARE complexation

result in an activated, fusion-ready state, and subsequent

mechanistic steps leading to fusion may well be SNARE-

independent, possibly even requiring disassembly (clear-

ance) of SNARE complexes in order to most fully facilitate

J.A. Szule, J.R. Coorssen / Biochimica et Biophysica Acta 1641 (2003) 121–135 129

the membrane merger steps. In this respect, the earliest

cartoons proposing a role for the SNAREs in exocytosis

may still be the most relevant [187]; a ‘black box’ still exists

between SNARE complex formation and the actual mem-

brane merger steps defining native fusion. Although a

mechanistic pathway to the fusion step has now been

proposed [81,82], this model requires testing in other

systems. A recent groundbreaking study clearly supports

the concept of a lipidic fusion pore, perhaps consistent with

the involvement of a proteolipid [188].

Strong evidence also exists for alternate SNARE func-

tions, in particular the regulation of Ca2 + and other ion

channels [189–197]; however, recent evidence suggests that

this represents an adapted modulatory function in mamma-

lian systems [198]. How does this, if at all, relate to the

proposed roles of SNARE complexation in fusion? One

possible explanation is that this is the actual function of the

SNAREs, to target and attach vesicles to appropriate ‘func-

tional’ sites, in part also regulating the channels and thereby

signalling pathways. It may be that accessory proteins

associated with the SNAREs (or the channel; [199,200])

mediate the actual fusion steps of exocytosis. Or, SNAREs

may simply have two (or more) separate but overlapping

roles in the exocytotic pathway. Alternatively, perhaps

SNARE complexes are artefacts of the techniques we

currently have at our disposal [201]. This is an intriguing

suggestion, implying that most SNARE complexes assayed

in vitro form as a result of sample aging and are therefore

not representative of functional native complexes. If this is

the case, it means that after tens of hours of aging in vitro,

CV SNAREs should be relegated to nonfunctional com-

plexes. Furthermore, as the same group also reports the

extremely high stability of SNARE complexes [202], CV

aged in vitro should be non-fusogenic. This is simply not the

case [36,76] [unpublished observations], again suggesting

that SNAREs do not function as the minimal fusogens of

native membranes.

Considering the CV studies, it is now reasonably certain

that the SNAREs (either alone or as complexes), having

carried out their critical upstream function(s), are not re-

quired during the Ca2 +-triggered steps of membrane fusion.

In a sense, the unfertilized urchin egg provides us with a

stage-specific ‘snapshot’ of these particular steps in the

exocytotic pathway. This would account for the speed of

the first fusion events within the population of f 15,000

fully docked and release-ready CV in an egg; fusion is at

least as fast as that measured in neuroendocrine cells, with

the lag-time after a rapid rise in [Ca2 +]free being < 10 ms

[151]. However, while eggs only undergo one such trig-

gered round of release, neurons and neuroendocrine cells are

capable of multiple high-rate rounds with appropriate stim-

uli. Cytoplasmic factors (e.g. NSF, a-SNAP and ATP)

causing disruption of cis SNARE complexes promote such

recycling by ensuring the availability of free SNAREs to

form inter-membrane complexes (e.g. priming in trans)

[145]. Thus, although we know that Ca2 +-triggered fusion

is extremely fast and seemingly optimized in neurons [150],

the implication is that the Ca2 +-triggered step(s) occurs after

SNARE action; SNAREs can promote the mechanism

without having an actual role in fusion itself. As protein

folding and conformational changes occur in the nanosec-

ond–microsecond time scale [203–205], numerous molec-

ular steps that are simply unresolved by current electro-

physiological or imaging methods could clearly occur

during the lag phase between Ca2 + entry and membrane

merger. Indeed, the time for membrane rearrangements and

merger via the stalk-pore pathway is estimated to be f 10

ns [183,206]. If Ca2 + has a role in clearing inter-membrane

SNARE complexes to promote the fusion mechanism

[36,76], this elevated [Ca2 +]free during strong stimulus

conditions would tend to promote complex disassembly.

Such a triggered loss of SNARE complexes appears to be

balanced upstream by Ca2 +-dependent SNARE complex

formation [94,207,208]. Late, fast Ca2 +-triggered disruption

of rapidly ‘zippered’ inter-membrane complexes would not

be detected in the majority of currently available assays due

to limited temporal resolution and the sheer magnitude of

concurrent reactions in (semi)intact preparations. To sum-

marize, the essential role of the SNAREs during the process

of exocytosis is undeniable, however, the evidence suggests

that they function at a pre-fusion rather than the fusion

stage.

In closing, we are reminded of Dr. G. Palade’s Nobel

Prize acceptance speech [209]:

‘‘A distinction should be made between agents directly

affecting fusion–fission and agents affecting the super-

imposed regulatory systems that activate and inactivate

the coupling between stimulation and secretion.’’

Acknowledgements

The authors wish to thank Gerald Zamponi, Roby Butt,

Joshua Kirshtein, Jeffery Lamb, Alana Luft and Sabine

Horn for discussions during the writing of this review.

J.R.C. notes many pleasant and productive conversations

with Paul Blank and Josh Zimmerberg during collaborations

on some of the work cited. J.R.C. acknowledges support of

the Alberta Heritage Foundation for Medical Research, the

Canadian Institutes of Health Research, the Heart and

Stroke Foundation of Canada and the Ruth Rannie

Memorial Fund (Faculty of Medicine, University of Cal-

gary). J.A.S. is the recipient of a Postgraduate Scholarship

Award from the Natural Sciences and Engineering Research

Council of Canada.

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